Biological Implications of a Stroke Therapy Based in Neuroglobin Hyaluronate Nanoparticles. Neuroprotective Role and Molecular Bases

Exogenous neuroprotective protein neuroglobin (Ngb) cannot cross the blood–brain barrier. To overcome this difficulty, we synthesized hyaluronate nanoparticles (NPs), able to deliver Ngb into the brain in an animal model of stroke (MCAO). These NPs effectively reached neurons, and were microscopically identified after 24 h of reperfusion. Compared to MCAO non-treated animals, those treated with Ngb-NPs showed survival rates up to 50% higher, and better neurological scores. Tissue damage improved with the treatment, but no changes in the infarct volume or in the oxidative/nitrosative values were detected. A proteomics approach (p-value < 0.02; fold change = 0.05) in the infarcted areas showed a total of 219 proteins that significantly changed their expression after stroke and treatment with Ngb-NPs. Of special interest, are proteins such as FBXO7 and NTRK2, which were downexpressed in stroke, but overexpressed after treatment with Ngb-NPs; and ATX2L, which was overexpressed only under the effect of Ngb. Interestingly, the proteins affected by the treatment with Ngb were involved in mitochondrial function and cell death, endocytosis, protein metabolism, cytoskeletal remodeling, or synaptic function, and in regenerative processes, such as dendritogenesis, neuritogenesis, or sinaptogenesis. Consequently, our pharmaceutical preparation may open new therapeutic scopes for stroke and possibly for other neurodegenerative pathologies.


Introduction
Stroke has become a global social issue that threatens health, and shortens life expectancy, affecting the quality of life worldwide [1]. Ischemic stroke, the most common type of stroke (80-85%), is caused by a blood clot or by the narrowing of a blood vessel, producing infarcts, generally in the territory irrigated by the middle cerebral artery, affecting the parietal cortex and striatum, which undergo histological and functional damages. In order to assess the severity of the infarct, a combination of magnetic resonance imaging (MRI) and neurological evaluation is used. In fact, the outcomes of imaging and pathological parameters are correlated to the results of learning and memory/mood studies [2].
Both core and penumbra zones can be distinguished within the infarcted brain, the last one being susceptible to recuperation only in cases of mild infarct severity, and when Figure 1 shows the mortality rates in the three experimental groups 24 h after surgery. As shown, the rates found in MCAO-Ngb animals fell down to half (25%) in relation to the MCAO group (50%).
Moreover, MCAO animals treated with Ngb (MCAO-Ngb) also improved their neurological outcomes, with a score of 4 points in relation to the non-treated group (MCAO), which reached near to 7 points in Bederson's scale ( Figure 2).

Ngb-NPs Cross the BBB, and are Endocytosed by Neurons
Immunofluorescence techniques and further confocal microscopy observations were performed to ascertain the penetration of the Ngb-NPs into the brain parenchyma, but mainly to determine its cellular localization at the site of the lesion, the parietal cortex, in MCAO animals 24 h after the onset of reperfusion.
Both empty-NPs ( Figure 3) and Ngb-NPs ( Figure 4) crossed the BBB, reached the brain parenchyma, and were endocytosed by the nervous cells. In addition, we have identified the NPs inside the neurons of the infarcted zone ( Figure 5). As shown in Figure 5 (A and D), most of the Ngb-NPs appeared inside the neurons as yellow granules in the overlaid confocal images, due the colocalization of red rhodamine-marked NPs with NeuN labelled with Cy-2 green fluorescence. As observed after 24 h of reperfusion, only few NPs

Ngb-NPs Cross the BBB, and are Endocytosed by Neurons
Immunofluorescence techniques and further confocal microscopy observations were performed to ascertain the penetration of the Ngb-NPs into the brain parenchyma, but mainly to determine its cellular localization at the site of the lesion, the parietal cortex, in MCAO animals 24 h after the onset of reperfusion.
Both empty-NPs ( Figure 3) and Ngb-NPs ( Figure 4) crossed the BBB, reached the brain parenchyma, and were endocytosed by the nervous cells. In addition, we have identified the NPs inside the neurons of the infarcted zone ( Figure 5). As shown in Figure 5 (A and D), most of the Ngb-NPs appeared inside the neurons as yellow granules in the overlaid confocal images, due the colocalization of red rhodamine-marked NPs with NeuN labelled with Cy-2 green fluorescence. As observed after 24 h of reperfusion, only few NPs were located outside the neurons. Cy5 fluorescence, digitally shown in grey, identified GFAP (astrocytes), which barely showed NPs at this reperfusion time.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 o were located outside the neurons. Cy5 fluorescence, digitally shown in grey, identif GFAP (astrocytes), which barely showed NPs at this reperfusion time.   were located outside the neurons. Cy5 fluorescence, digitally shown in grey, identified GFAP (astrocytes), which barely showed NPs at this reperfusion time.           The determination of the oxidative (TBARS) and nitrosative stress (NOx) in the tissues of MCAO and MCAO-Ngb animals also showed non-significant changes in relation to sham animals ( Figure 8).

Ngb-NPs Treatment does not Affect the Infarct Size or the Oxidative/Nitrosative Stress but Improves the Histological Outcomes
Nevertheless, the microscopic analysis of the histological sections from the damaged area stained with cresyl-violet showed that MCAO-Ngb individuals exhibited less histological damage than the MCAO animals, where scarce cell bodies could be found ( Figure  9). The determination of the oxidative (TBARS) and nitrosative stress (NOx) in the tissues of MCAO and MCAO-Ngb animals also showed non-significant changes in relation to sham animals ( Figure 8).  The determination of the oxidative (TBARS) and nitrosative stress (NOx) in the tissues of MCAO and MCAO-Ngb animals also showed non-significant changes in relation to sham animals ( Figure 8).
Nevertheless, the microscopic analysis of the histological sections from the damaged area stained with cresyl-violet showed that MCAO-Ngb individuals exhibited less histological damage than the MCAO animals, where scarce cell bodies could be found ( Figure  9).  Nevertheless, the microscopic analysis of the histological sections from the damaged area stained with cresyl-violet showed that MCAO-Ngb individuals exhibited less histological damage than the MCAO animals, where scarce cell bodies could be found ( Figure 9). The total amount of proteins initially identified and quantified in the 12 samples studied was of 4077 proteins (expressed by MaxQuant as "Protein groups"). This initial set of proteins underwent several steps of filtering using the Bioconductor package DEP, as previously explained. The statistical analysis performed using the DEP package provides an integrated analysis workflow for robust and reproducible analysis of mass spectrometry proteomics data for differential protein expression or differential enrichment. Specifically, the 4077 proteins obtained (after the MaxQuant analysis) became 3984 when contaminants and reverse (decoy) sequences were removed. Then, proteins with only one peptide were also filtered, obtaining 3298 proteins. Finally, this number became 2573 (Supplementary data: Differential analysis results annotated), considering only those proteins present in at least one sample in each of the three studied conditions, grouped as sham, MCAO, and MCAO-Ngb.

Protein Identification and Quantification
The total amount of proteins initially identified and quantified in the 12 sam studied was of 4077 proteins (expressed by MaxQuant as "Protein groups"). This i set of proteins underwent several steps of filtering using the Bioconductor package as previously explained. The statistical analysis performed using the DEP package vides an integrated analysis workflow for robust and reproducible analysis of mass trometry proteomics data for differential protein expression or differential enrichm Specifically, the 4077 proteins obtained (after the MaxQuant analysis) became 3984 w contaminants and reverse (decoy) sequences were removed. Then, proteins with only peptide were also filtered, obtaining 3298 proteins. Finally, this number became (Supplementary data: Differential analysis results annotated), considering only those teins present in at least one sample in each of the three studied conditions, group sham, MCAO, and MCAO-Ngb.

Quality Control and Differential Analysis
A first approach for quality control, based on filtering the proteins found in all ples from the three groups, quantified almost 2000 proteins, of which the 500 most v ble were chosen to perform a principal components analysis (PCA). As shown in F 10, the PCA grouped the different samples according to their corresponding cond

Quality Control and Differential Analysis
A first approach for quality control, based on filtering the proteins found in all samples from the three groups, quantified almost 2000 proteins, of which the 500 most variable were chosen to perform a principal components analysis (PCA). As shown in Figure 10, the PCA grouped the different samples according to their corresponding condition, i.e., sham, MCAO, or MCAO-Ngb groups; interestingly, the MCAO-Ngb animals were clustered closer to the sham control group than to the MCAO group.
A second approach for quality control was performed under a p-value of 0.02 and a Log2 Fold change of 0.5 as cut-offs. These thresholds are reliable enough [49] to identify the most significant changes in the expression of proteins in our model induced by the stroke and the treatment with Ngb. Figure 11 shows the volcano plots obtained in each of the three comparisons included in the study, where several proteins presented a clear over-or underexpression depending on stroke and/or treatment with Ngb. Specifically, when comparing MCAO vs. sham, proteins FBXO7, WIPI2, NTRK2, A0A0G2JY03, and ITGB8 appeared clearly underexpressed, whereas MAP1a and CPQ were overexpressed, indicating a clear effect of the stroke (MCAO injury) on the individuals. In the second comparison, MCAO-Ngb vs. sham, proteins PPP2r2c, WIPI2, and LRRC8d were underexpressed, whereas ATXN2l, MAP1a, and TBC1d10b appeared overexpressed. Finally, in the comparison between MCAO-Ngb and MCAO, which demonstrates the effects of the treatment with Ngb-NPs in animals submitted to stroke, the most evident changes involved overexpression of ATXN2l, FBXO7, and NTRK2. These results show that the treatment with Ngb-NPs induces expression of new proteins, or even reverses the expression induced by the effect of the stroke. i.e., sham, MCAO, or MCAO-Ngb groups; interestingly, the MCAO-Ngb animals w clustered closer to the sham control group than to the MCAO group. A second approach for quality control was performed under a p-value of 0.02 an Log2 Fold change of 0.5 as cut-offs. These thresholds are reliable enough [49] to iden the most significant changes in the expression of proteins in our model induced by stroke and the treatment with Ngb. Figure 11 shows the volcano plots obtained in eac the three comparisons included in the study, where several proteins presented a c over-or underexpression depending on stroke and/or treatment with Ngb. Specifica when comparing MCAO vs. sham, proteins FBXO7, WIPI2, NTRK2, A0A0G2JY03, ITGB8 appeared clearly underexpressed, whereas MAP1a and CPQ were overexpres indicating a clear effect of the stroke (MCAO injury) on the individuals. In the sec comparison, MCAO-Ngb vs. sham, proteins PPP2r2c, WIPI2, and LRRC8d were unde pressed, whereas ATXN2l, MAP1a, and TBC1d10b appeared overexpressed. Finally the comparison between MCAO-Ngb and MCAO, which demonstrates the effects of treatment with Ngb-NPs in animals submitted to stroke, the most evident changes volved overexpression of ATXN2l, FBXO7, and NTRK2. These results show that the tr ment with Ngb-NPs induces expression of new proteins, or even reverses the expres induced by the effect of the stroke.

Hierarchical Clustering
A hierarchical clustering study has been performed using proteins that showed expression changes in at least one of the comparisons among the three groups under a p-value lower than 0.02. This filter produced a set of 219 proteins affected. For each protein and sample, the normalized intensity was used to make a matrix of 219 × 12. The matrix was scaled obtaining a maximum of +2.42, and a minimum of −2.86. This matrix was used to perform a hierarchical cluster using the pheatmap package, where both rows (proteins) and columns (samples) have been clustered, i.e., they have freely organized themselves using the individual intensities of the matrix (unsupervised machine learning both for proteins and samples). The optimal number of clusters has been obtained using the find_k function from the dendextend R package. The proteins were grouped in five clusters that accurately describe how protein intensities have been organized in different categories that can be represented in a hierarchical cluster ( Figure 12). In this representation, both proteins and genes have been used to annotate the rows; the columns are represented by the 12 samples used in the study.
The three conditions: MCAO-Ngb, sham, and MCAO (in this order) have been used to annotate the samples, whereas the five main clusters are shown as cluster 1 to cluster 5. Different general patterns of intensities are found for each of the clusters and sample types (activation is represented in red, and underexpression in blue). Once again, it is clear that the different expression intensities of these 219 proteins are able to perfectly classify the 12 samples into the three conditions (groups) of our study. The hierarchical cluster also shows the trends of the changes in the protein expression for the five clusters (1 to 5) in the MCAO-Ngb, sham, and MCAO groups, respectively. Samples belonging to the group of the stroke condition (MCAO) show, in general, more variability than the other two conditions (sham and MCAO-Ngb), especially in cluster 1 and cluster 4, which could point to a high heterogeneity in the biological reaction to a stroke event.

GO and Pathways Enrichment
Two enrichment studies have been performed. First, each of the five clusters of the hierarchical cluster study was analyzed separately. Second, all the proteins were analyzed together (the proteins in this study included 517 proteins with a p-value lower than 0.05 in at least one of the three comparisons); the enrichment was performed with STRING database, version 11.0, and using Rattus norvegicus species.

Individual Enrichments in Each of the Five Clusters
Five different experiments of enrichment, using gene ontology (GO), biological function category (Gene Ontology Resource), and KEGG pathways (KEGG PATHWAY Database) have been performed using the proteins in each of the clusters shown in the hierarchical cluster ( Figure 12). Results are displayed in Table 1, where, as exposed, only clusters 1 to 4 showed remarkable results.
Actin binding has been found as one of the biological functions most affected, as shown in clusters 1 and 2, with three and four genes involved, respectively. One of these genes in cluster 2 corresponds to Map1a, previously found as overexpressed due to stroke. Another biological function affected is "GTPase binding", grouped in cluster 3 with five genes involved; and "Ras GTPase binding" in cluster 3 and 4 with four genes involved in both clusters. Special attention should be given to the KEGG pathways "endocytosis" and "protein processing in endoplasmic reticulum", both concerning cluster 2, and showing a total of four and three dysregulated genes, respectively.

Ngb_3
Ngb_2  A GO enrichment (only for biological function) and pathway enrichment (for KEGG pathways) have been performed using the 517 proteins that showed a p-value lower than 0.05 in at least one of the three comparisons performed (MCAO vs. sham; MCAO-Ngb vs. sham; MCAO-Ngb vs. MCAO). The results can be found in Table 2. A total of 113 terms show some enrichment (FDR<0.05); 75 of them are biological function terms, and the other 37 are KEGG pathways. Terms with a background (complete set of genes in the category) higher than 300 have been removed from this list. The row corresponding to the KEGG pathway "endocytosis", with 28 proteins mapped, has been split. This global enrichment endorses the results obtained in the individual enrichment previously performed. Thus, the most altered biological functions are related with cytoskeletal protein interactions (i.e., "actin" or "microtubule bindings"), and "GTP signaling" and "energy metabolism" (i.e., GTPase, GTP, GDP, or Ras GTPase bindings). In relation to KEGG pathways, the most significant ones were "endocytosis", as well as "protein synthesis, processing and degradation".

Pathway Analysis
The pathway analysis using Kegg pathways has been implemented with the Bioconductor package Pathview (pathview). Only proteins with a p-value lower than 0.05 in at least one of the three comparisons have been used. The pathways selected are those that have shown significant enrichment in the previous section (Table 2), and showed some relationship with the matter under study. Figure 13 shows the "endocytosis pathway" as an example of one the most affected pathways, where each protein area is divided into three sectors: the one to the right shows the fold change for the MCAO vs. sham, the one in the center for the MCAO-Ngb vs. sham, and the one in the right for the MCAO-Ngb vs. MCAO. Red color means overexpression, and green means underexpression. This last analysis ratifies that the treatment with Ngb-NPs is involved in some transcendent biological processes, such as endocytosis, protein metabolism, synaptic function, and neurotransmission (synthesis of neurotransmitters-GABA, serotonin, glutamic acid-or synaptic vesicles traffic), or even in molecular pathways recurrent in some neurodegenerative diseases, such as Huntington's, Parkinson's, or Alzheimer's.

Discussion
The present research demonstrates that, when injected systemically, Ngb-NPs can cross the BBB, and be endocytosed by neurons of the infarcted area in a rat model of stroke (MCAO). Once there, Ngb exerts its neuroprotective action only after 24 h of reperfusion. It has been proved that the animals submitted to stroke, and treated with Ngb-NPs have higher survival rates and better behavioral scores than the MCAO animals. The histological status also improved with the treatment, although at this early time of reperfusion, no changes neither in the infarct volume nor in the oxidative or nitrosative stress assessments were detected in the damaged infarcted area. Finally, the proteomic changes induced by stroke and the Ngb-NPs treatment has been analyzed with the aim of unravelling the molecular pathways by which Ngb exerts its neuroprotective action. These changes affect some biological processes, such as endocytosis, cytoskeletal remodeling, or some metabolic routes, all of them related to damage, and restorative mechanisms that are involved in dendritogenesis, neuritogenesis, or sinaptogenesis.
Currently, there are multiple restorative therapies against stroke addressed to avoid some processes of the ischemic cascade, such as oxidative/nitrosative stresses, or glutamate excitotoxicity or Ca 2+ overload, which may finally may trigger the activation of necrosis and apoptosis processes [50]. These therapies range from the most conventional (anticoagulants, decompressive surgery, or hypothermia) to the most recent (monoclonal antibodies, cell-based therapy, or even robotics stimulation) [51,52]. Additionally, pharmacological treatments using growth factors (BDNF, EGF plus EPO, and hCG plus EPO), specific neurotransmitters (oxalate, glutamate), or nitric oxide (NO) synthase inhibitors have been tested [53][54][55]. Interestingly, the administration of nutritional supplements in the diet is gradually reaching greater interest [56,57]. Other new promising therapies are based on emergent neuroprotectants, such as antioxidant enzymes like Peroxiredoxin-6 [58], or even other proteins with a wide neuroprotective capacity, such as the protein Ngb [13]. However, the therapeutic usage of this protein is still limited because of its difficulty to cross the BBB. To avoid this handicap, different types of drug delivery systems have been developed, such as the use of cell-penetrating peptides (CPP). In this vein, we have synthesized different types of hyaluronate nanoparticles optimizing their size and electrical charge, making them suitable to avoid the immune system, and cross the BBB after stroke [47]. Then, we selected the best typology of these NPs to transport an adequate dose of rat recombinant Ngb from the bloodstream to the cerebral parenchyma. This new pharmaceutical preparation was injected immediately after the stroke, and was delivered into the ischemic cerebral area as early as 2 h after stroke, and was still detectable after 24 h of reperfusion [48]. In order to ascertain the biological implications and neuroprotectant ability of our pharmaceutical preparation of Ngb, our first objective here was to demonstrate that the Ngb-NPs could be endocytosed by neurons. This approach has been confirmed using immunofluorescence and confocal microscopy; in fact, the confocal images obtained have allowed us to assert that Ngb-NPs have not only crossed the BBB, but have finally been captured by neurons, as demonstrated with the colocalization of such Ngb-NPs with the specific neuronal protein, NeuN [59]. Actually, we do not know the exact pathway followed by the Ngb-NPs from blood to neurons, and although it has been described that the BBB can be disrupted transiently [38,39] or continuously [40] during stroke, some authors have also reported that Ngb does not efficiently cross the BBB in ischemic mice [41]. Therefore, the Ngb-NP should go through the many cellular layers of the BBB by transcytosis mechanisms until they reach the neurons. Our microscopic images show that, after 24 h of reperfusion, the neurons have already captured most of the NPs systemically injected in the vascular network; these NPs appear clustered in their cytoplasm, although a few of them are also located within other nervous cells. Hence, the specific cytoskeletal protein of astrocytes GFAP barely shows colocalization with our NPs. Interestingly, it has been reported that isolated cultured astrocytes express and release endogen Ngb towards neurons packed inside of exosomes [60]. Considering the above, it would be possible to speculate that astrocytes from the BBB could release our NPs using this same pathway, but also, this transcytosis would be over after 24 h of the onset of the reperfusion.
Another important subject of our research concerns the fact that the MCAO-Ngb animals doubled their survival rates, and showed improved neurologic scores compared to non-treated animals. There are many reports that document the protective role of Ngb in stroke and other neurodegenerative diseases [13], but only a few of them have analyzed survival rates and/or neurological outcomes after stroke. Xue and collaborators [61] found that high serum levels of endogen Ngb, whose peak is reached 72 h after ischemic stroke, correlated significantly with both infarct size and admission NIHSS score in humans. A study that combined overexpression of Ngb and an inhibitor of the c-jun N-terminal kinase (JNK), carried out in male spontaneously hypertensive prone stroke rats, reported that this treatment reduced the infarct size, and improved the neurologic outcome 14 days after transient MCAO (tMCAO) [62]. Our research shows that as early as 24 h after stroke, the survival rates and the neurological scores improved with the Ngb-NPs treatment; on the other hand, no changes were detected in the infarct size, or in the oxidative and nitrosative stresses statuses. Thus, we hypothesize that 24 h of reperfusion may be too soon to observe these types of changes, despite the findings of better outcomes in the tissue status. Certainly, new studies with longer reperfusion times and even higher Ngb dosage should be conducted for a better assessment of the therapeutic effects of Ngb.
Regarding the most significant proteomic changes induced by Ngb-NPs in stroke animals, they involved proteins featuring some key pathways, such as endocytosis and vesicular traffic, cytoskeletal remodeling, metabolism, or synaptic transmission. All these pathways are highly related to neurodegenerative tissue damage and regeneration. Interestingly, a total of at least 219 proteins from these pathways underwent noticeable expression changes, although we have focused on the most significant proteins, previously identified in the volcano plots. Particularly, the ischemic lesion induced the underexpression of FBXO7 and NTRK2, although when stroke animals were treated with Ngb-NPs, both proteins underwent a strong recovery and even overexpression. FBXO7 (F-box only protein 7) corresponds to an E3-ubiquitin-protein ligase that participates in the proteasomal degradation of target proteins involved in mitophagy and negative regulation of stress cellular death. At the moment, FBXO7 has been related to Parkinson's disease [63], although to the best of our knowledge, it has not been directly involved in stroke. In fact, FBXO7 mediates the proteasomal degradation of UXT-V2, causing the inhibition of the NF-κB signaling pathway, suggesting that FBXO7 plays roles in mitochondrial transport and Wnt signaling regulation [64], a pathway highly related to neurogenesis [24]. NTRK2 (high affinity nerve growth factor receptor) is a receptor of BDNF that is also involved in neurogenesis and neuronal plasticity; this protein has been related to stroke throughout the pathway BDNF-TrKBPI3K/Akt. Therefore, Ngb could activate the PI3K/Akt pathway by increasing the BDNF levels, and thus, enhance synaptic plasticity, and repair processes of damaged neurons [65].
Other proteins, such as MAP1a and CPQ, are overexpressed due to MCAO lesions, and remain elevated even after the treatment with Ngb-NPs. MAP1a (microtubule-associated protein 1a) is a structural protein mainly located in the dendrites of adult neurons involved in the filamentous cross-bridging between microtubules and other skeletal elements. Thus, this protein controls the microtubule assembly [66], but can also interact with other cellular components, including filamentous actin and signaling proteins [67]. Interestingly, the activity of MAP1a is controlled by upstream signaling mechanisms through the mitogenactivated protein (MAP) kinase, suggesting that its dendritic remodeling activity depends on this pathway. The fact that the treatment with Ngb-NPs maintains MAP1a levels elevated after induction by stroke, is probably to favor its restorative activity repairing the dendrites of neurons damaged by the ischemic stroke. Regarding CPQ, a carboxypeptidase that may play an important role in the hydrolysis of circulating peptides, it also follows the same expression pattern as MAP1a. To our knowledge, it has not been previously detected in a stroke-damaged brain, but our results document that MCAO induces its expression in both MCAO and MCAO-Ngb animals.
Another downregulated protein is WIPI2. This protein (WD repeat domain phosphoin ositide-interacting protein 2) is involved in autophagy during mitosis, via CUL4-RING ubiquitin ligases-mediated WIPI2 polyubiquitination and proteasomal degradation [68]. In fact, WIPI2 protein level, regulated by MTORC1 and HUWE1 (an E3 ubiquitin ligase), plays a determinant role in cellular autophagy intensity, suggesting that the quantity control exerted by WIPI2 affects autophagy-related physiopathological processes [69]. Our results showed a decrease of this protein after stroke, remaining underexpressed with Ngb treatment; consequently, stroke could regulate the intensity of autophagy throughout the underexpression of this protein.
A specific feature of the Ngb-NPs treatment is the overexpression of ATX2L. This protein is involved in the assembly of the stress granules (formed in response to diverse types of stresses), and in the cytoplasmic mRNA processing bodies [70]. Moreover, this protein contributes to the cytoskeletal reorganization, and to the recovery of the mitochondrial function through its role in different metabolic pathways. In fact, this protein has also been related to the "Wnt signaling pathway" [71], which, as was previously mentioned, is involved in neurogenesis.
On the other hand, our proteomic approaches also show important expression changes in up to 28 different proteins involved in endocytosis, predicting that stroke and the later treatment with Ngb may interfere with the vesicular traffic. One of these proteins is the Ras-related protein RAB-4A, which is underexpressed, but only due to Ngb-NPs treatment. This is a small GTPase associated-protein with endocytic compartments and a key regulator of early endosomes recycling [72]. In addition, it cycles between an active GTP-bound and an inactive GDP-bound state, being associated with endosomal vesicular traffic through "VEGFR2 signaling", a pathway related to angiogenesis [73]. What is more, this pathway is associated with the protein, endophilin (ENDO), which is overexpressed either in MCAO and MCAO-Ngb animals. Endophilin was identified as a component of clathrin-mediated endocytosis [74], and its deficiency reduces VEGFR2 internalization, and, therefore, regulates angiogenesis. EH-domain-containing 4 (EHD4) is also a protein of the endocytic network that is downregulated both by stroke and treatment with Ngb. This protein controls early endosome trafficking, promotes the recycling of internalized transmembrane proteins [75], and is related to the pathways underlying the axonal transport as well [76]. Another important protein involved in the endocytic pathway is the neural Wiskott-Aldrich syndrome protein (N-WASP or WASL), an actin nucleation factor responsible for the polymerization of branched actin filaments that underwent upregulation by MCAO, but later downregulation due to the treatment with Ngb. The activity of this protein is essential for myelin membrane wrapping and, therefore, for neuritogenesis [77]. Additionally, N-WASP takes part in the development of dendritic spines and synapses [78].
In the light of the important number of endocytic proteins entailing pathways such as dendritogenesis, neuritogenesis, or even sinaptogenesis, it can be inferred that both the MCAO injury and the treatment with Ngb-NPs are the main inductors of the regulation of these repairing mechanisms. Certainly, the displacement of the NPs (empty or linked to Ngb) from blood towards neurons could also influence the endocytic pathway, but this should have been equally observed in the three groups, as all of them received the same dose of hyaluronate NPs.
In consequence, Ngb seems to induce changes in the expression of a myriad of proteins associated to a variety of biological processes, all of them contributing to reveal the molecular bases of its neuroprotective role, mainly centered in avoiding neuronal death, and triggering reparation mechanisms.

Biosynthesis of Ngb-NPs
By means of ionic gelation methods, we previously designed different types of hyaluronate NPs coated with chitosan and glycerol tripalmitin. Final size (245 nm) and surface charge (about 32 mV) of the NPs were adequate for their use as therapeutic tools. These NP were able to cross through the BBB, and to be endocytosed by nervous cells [47], even after being attached to rat-recombinant Ngb, previously prepared in our lab [48]. These Ngb-NPs were evaluated in size, ζ-potential, encapsulation degree, and in vitro release; in addition, their kinetic liberation was assessed as well. The results showed that the formulation was highly compatible with its pharmaceutical use, and that it could act as a delivery system to transport Ngb throughout the blood towards the brain parenchyma. A comprehensive analysis of the composition and characteristics of these Ngb-NPs has been previously published by our group [47,48].

Study Design
The animals (n = 94) were homogeneously distributed into three different experimental groups: (1) sham-operated rats injected with empty NPs (sham), used as control; (2) rats submitted to MCAO and injected with empty NPs (MCAO); and (3) rats submitted MCAO and injected with Ngb-NPs (MCAO-Ngb). After surgery, with the mortality rates taken into account (Figure 1), the number of surviving animals used for all analyses and determinations yielded an n = 64.
NPs, either empty or bound to Ngb (2.15 mg/mL) [48], were systemically injected via the lateral tail vein immediately after the surgery at the onset of reperfusion. The total number of animals per group was distributed according to each of the techniques listed, minimizing the use of animals, but guaranteeing an adequate "n" per group to ensure an appropriate statistical confidence.

Stroke Model
Transient focal ischemia was induced by occlusion of the middle cerebral artery as previously described [79,80], with slight modifications [47,48]. This model of brain ischemia is considered one of the best models to mimic human ischemic stroke [81], representing an appropriate in vivo procedure to define the protective role of Ngb-NP against stroke.
Briefly, the middle cerebral artery is occluded using a nylon filament suture with a 3-4 mm coating (Doccol Corporation, Redlands, CA, USA), inserted through the right common carotid artery, and advanced until the origin of the middle cerebral artery. A laser-Doppler flow probe (tip diameter 1 mm) attached to a flowmeter (moorVMS-LDF1, Moor Instruments, Axminster, UK) is located over the thinned skull in the MCAO territory (4 mm lateral to bregma) to obtain a continuous measure of relative cerebral brain flow during the surgery. Only animals with a cerebral blood flow reduction over 60% were included in the study. After surgery, anesthesia was discontinued, and rats were returned to their home cages. Animals in the three groups studied were sacrificed 24 h after MCAO procedure.

Mortality Rate and Neurological Outcomes
The mortality rate was assessed by calculating the percentage of animals in each experimental group that survived the MCAO intervention, but died within the 24 h of reperfusion period.
A double-blind evaluation of the neurological outcomes induced by MCAO and reperfusion was carried out in all animals immediately before neuroimaging or sacrifice following Bederson's neurological scoring [82]. This evaluation was carried out in the animal facilities of the University of Jaén, which are equipped with a specific module for animal behavioral assessment. More specifically, we measured the neurological deficits according to the scoring system depicted in Table 3.

MRI Neuroimage Study
In order to document the extent of the ischemic damage, an MRI was performed prior to euthanasia on a 9.4T Biospec Avance III small animal MR system (Bruker BioSpin, Ettlingen, Germany) equipped with 400 mT/m field gradients, and operated by a Paravision 6.0 software platform. A circular polarized volume resonator was used for signal transmission, and an actively decoupled rat brain quadrature surface coil for signal reception (Bruker BioSpin, Ettlingen, Germany). During the experiment, isoflurane (Abbott Laboratories, Madrid, Spain) was used for anesthesia (3.5% for induction for 2 min, and approximately 1.0 % for maintenance in oxygen at a flow rate of 1-1.5 mL/min). Respiration of the animal was monitored using a pneumatic cushion respiratory monitoring system (Small Animal Instruments Inc., Stony Brook, NY, USA). Animals were placed on a water-heated rat cradle, and the head was immobilized using ear and tooth bars. Body temperature was measured using a rectal thermometer, and maintained at 37 • C. First, fast gradient echo images were acquired as reference images. High resolution T2-weighted MR images were acquired using a RARE (rapid acquisition with relaxation enhancement) sequence with the following parameters: TR = 2500, TE = 33 or 60 ms, in-plane resolution = 83 m, slice thickness = 1 mm, and ETL = 8. T2 parametric maps were also acquired using a multi-echo spin echo sequence (32 echoes and 7 ms inter-echo spacing). The volumes of the infarcts shown in the T2 images were obtained using Image J software. These experiments were performed at the Andalusian Centre for Nanomedicine and Biotechnology (BIONAND).

Histological Studies
Animals were perfused through the left ventricle with pH 7.4, 0.01 M phosphatebuffered saline (PBS), followed by 300 mL of 10% formalin. Brains were removed and post-fixed for a further 4 h in the same fixative at room temperature as previously published (Blanco et al., 2020). Then, the brains were cryoprotected for 48 h by immersion in 0.1 M phosphate buffer (PB) containing 30% sucrose at 4 • C, and embedded in O.C.T. medium. Serial rostro caudal sections (30 µm thick) were obtained using a cryostat (Leica CM1950, Leica Microsistemas S.L.U., L'Hospitalet de Llobregat, Spain). Serial sections were submitted to different histological techniques.

Cresyl-Violet (Nissl) Staining
This technique was used in order to evaluate the histological damage within the brain tissue. Briefly, sections were stained using a 0.1% solution of cresyl-violet, dehydrated, and mounted with DPX.
In all the confocal images, rhodamine-stained NPs were visualized in red (excitation 507 nm, emission 525 nm), and the cell nuclei were visualized in blue due to DAPI staining (excitation 358 nm, emission 529 nm). The other markers were visualized with different immunofluorescence dyes conveniently chosen according to the corresponding research purpose. Thus, to show colocalization between NPs and Ngb, Ngb was marked with Cy2 (excitation 492 nm, emission 510 nm), and observed in green. To detect the cellular localization of the NPs in the brain parenchyma, NeuN was marked with green fluorescent-Cy2 (excitation 492 nm, emission 510 nm), and GFAP was marked with Cy5 (excitation 650 nm, emission 670 nm), digitally shown in grey for better understanding.
To ensure that the signal detected in each channel was not due to autofluorescence, TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Burlingame, CA, USA) and control samples of rats not injected with NPs were used.

Oxidative and Nitrosative Stresses Determinations
The scavenging properties of Ngb were assessed by determining the oxidative/nitrosative statuses. Lipid peroxidation and NO production were measured using the method described by Buege and Aust [83] (thiobarbituric acid reactive substances, TBARS), and by means of indirect quantification of NO production by measuring nitrate/nitrite and S-nitrose compounds (NOA ™ 280i, Sievers Instruments Inc.-Zysense, Frederick, CO, USA), respectively, in ischemic brain tissues of the infarcted hemisphere from animals of each experimental group.

Sample Preparation and Mass Spectrometry Analysis
A total of 12 samples, 4 per condition (sham, MCAO, and MCAO-Ngb), were included in the proteomic studies. First, samples were incubated with 7 M urea, 2 M Thiourea, 4% CHAPS, and 5 mM DTT. Proteins were extracted using a Precellys tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Then, proteins were digested following the filter-aided FASP protocol described by Wisniewski [84] with minor modifications. Trypsin was added in a trypsin:protein ratio of 1:50, and the mixture was incubated overnight at 37 • C, dried out in a RVC2 25 speedvac concentrator (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), and resuspended in 0.1% formic acid (FA). Peptides were desalted and resuspended in 0.1% FA using C18 stage tips (Merck Millipore, Burlington, MA, USA). Later, samples were analyzed in a novel hybrid trapped ion mobility spectrometry, quadruple time of flight mass spectrometer (timsTOF Pro with PASEF, Bruker, Billerica, MA, USA), coupled online to a nanoElute liquid chromatography (Bruker, Billerica, MA, USA). This mass spectrometer takes advantage of a novel scan mode termed parallel accumulation-serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity [85], and has been proven to provide outstanding analytical speed and sensibility for proteomics analyses [86]. The sample (200 ng) was directly loaded in a 15 cm Bruker nanoelute FIFTEEN C18 analytical column (Bruker, Billerica, MA, USA), and resolved at 400 nl/min with a 100 min gradient. The column was heated to 50 • C using an oven. These experiments were performed at the Centre for Cooperative Research in Biosciences (CIC bioGUNE).

Bioinformatics Analysis
The raw files obtained by the MS/MS instrument were analyzed using MaxQuant (https://www.nature.com/articles/s41592-018-0018-y (accessed on 30 November 2021)) in a Linux cluster.
The statistical analysis includes comparisons two to two, among the three conditions. Specifically: MCAO vs. sham, MCAO-Ngb vs. MCAO, and MCAO-Ngb vs. sham. All these comparisons showed the differential protein expression induced either by stroke, by Ngb treatment, or by both.
For ordinary differential analysis and missing data imputation, we used the Bioconductor package DEP (https://www.biorxiv.org/content/10.1101/668863v1.full (accessed on 30 November 2021)). This package provides an integrated analysis workflow for robust and reproducible analysis of mass spectrometry proteomics data for differential protein expression or differential enrichment. Thus, the quantitation matrix performed using DEP allowed us to obtain the different trends for proteins along the three different experimental groups. Briefly, the steps followed using DEP software involved a series of filters to detect only the most variable proteins. In particular, we used the filter_missval DEP function: with the option "thr = 0", where only proteins that were identified in all replicates of at least one condition were selected. After data normalization (variance stabilizing transformation), missing data imputation was performed using random draws from a Gaussian distribution centered around a q-value = 0.01. Finally, after obtaining the differential expression analysis for the three comparisons under study, a p-value under 0.02 and Log2 fold change above 0.5 were used as threshold.
Then, the data were analyzed using different statistical approaches. First, an unsupervised classification algorithm, hierarchical clustering and k-means clustering, was used, looking for groups of proteins acting in a coordinated way. Second, an analysis of gene ontology (GO) and pathways enrichment using STRING (https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC6323986/ (accessed on 30 November 2021)) was performed. And third, a pathway projection of the proteins of interest was performed (KEGG, Reactome).

Statistical Analysis
The statistical analysis for the proteomics studies has been performed, as just mentioned, using the MaxQuant and DEP package. To analyze the other parameters studied (i.e., neurological outcomes, infarct volumes, TBARS, and NOx), the SPSS package (SPSS Inc., Chicago, IL, USA) was used. Data are expressed as means ± SD. Statistical comparisons were made using the Levene test to determine the homoscedasticity, and the Student's t-test to compare the means among the two experimental groups. Statistical significance was set at p < 0.05.

Conclusions
Ngb-NPs systemically injected in a rat model of stroke (MCAO) can cross the BBB, and are endocytosed by neurons of the infarcted area, triggering a neuroprotective action after 24 h of reperfusion.
MCAO-Ngb animals showed higher survival rates, as well as better behavioral scores and improved histological status, although no changes either in the infarct volume or in the oxidative and nitrosative stress assessments were found with the treatment.
The proteomic changes induced by stroke, and the treatment with Ngb-NPs indicated changes in some biological processes, such as endocytosis, cytoskeletal remodeling, or in some metabolic routes; all of them related to damage and restorative mechanisms involved in dendritogenesis, neuritogenesis, or sinaptogenesis.